Blood type checking reagent containing lectin

ABSTRACT

Lectin for checking a blood type is extracted from seeds of bitter gourd, balsam pear, or  Momordica charantia . A blood type checking reagent includes the lectin. The lectin extracted from seeds of bitter gourd preferably has a molecular weight between 100,000 and 170,000 measured with poly-acrylamide gel electrophoresis in existence of sodium dodecyl sulfate (SDS-PAGE). A method of checking a blood type includes the steps of: extracting lectin from seeds of bitter gourd, balsam pear, or  Momordica charantia ; preparing a blood type checking reagent containing the lectin; and checking a blood type using the blood type checking reagent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional application of the prior application Ser. No.11/253,667 filed Oct. 20, 2005, pending. The disclosure of JapanesePatent Application No. 2004-315366, filed on Oct. 29, 2004, isincorporated in the application by reference.

BACKGROUND OF THE INVENTION AND RELATED ART STATEMENT

The present invention relates to a blood type checking reagentcontaining a lectin. The blood type checking reagent exhibits specificcohesion relative to H-antigen on a red blood cell membrane or anerythrocyte membrane. Accordingly, it is possible to accurately andquickly determine a blood type even from a small bloodstain.

In general, lectin extracted from a plant includes anti-A1 lectin (fromDolicos biflorus) and anti-H lectin (from Ulex europaeus). The anti-Hlectin extracted from Ulex europaeus has been used for, for example,determining a blood type. Anti-H lectin with improved activity is calledH-antigen lectin strong, and suitable for checking a blood type of atrace amount of body fluid and a hair (dissociation test). The anti-Hlectin strong has agglutinin tilter four times larger than that ofconventional anti-H lectin. Accordingly, it is possible to determine ablood type of a small amount of sample (refer to Non-patent Reference 1and Non-patent Reference 2). The following table shows comparison ofagglutinin tilter between the anti-H lectin strong and the conventionalanti-H lectin.

Agglutinin tilter* Anti-H lectin 2⁶ (64)  Anti-H lectin strong 2⁸ (256)*Measured with continuous dilution method (2% O-type human erythrocyteagglutinin tilter PBS)

Further, lectin may be extracted from animal such as loach, especiallyfrom an egg thereof. The lectin extracted from loach has a molecularweight of 15,000 to 50,000, and is used for determining a blood type(refer to Patent Reference 1).

Non-patent Reference 1: Japanese Society of Laboratory Medicine LibraryXII (1996) Non-patent Reference 2: Product Report No. 105 (SeikagakuCorporation) Patent Reference 1: Japanese Patent Publication No.11-083862

The conventional method of checking a blood type has the followingdisadvantages. First, it is difficult to determine a blood type sampledfrom a bloodstain (dissociation test) using the anti-H lectin extractedfrom Ulex europaeus. Second, it is difficult to obtain raw materials forproducing the blood type checking reagent using the conventional anti-Hlectin. Further, it is necessary to use a large amount of raw materials,thereby increasing cost. Third, the agglutinin tilter of the lectinextracted from Ulex europaeus largely depends on a raw material (seed),and it is difficult to obtain constant agglutinin tilter within lot.Fourth, it is difficult to detect H-antigen using the lectin extractedfrom loach.

In view of the problems described above, an object of the presentinvention is to provide lectin for checking a blood type and a bloodtype checking reagent containing the lectin, in which it is possible toaccurately and quickly determine a blood type even from a smallbloodstain. Another object of the present invention is to provide amethod of checking a blood type using the blood type checking reagentcontaining the lectin.

Further objects and advantages of the invention will be apparent fromthe following description of the invention.

SUMMARY OF THE INVENTION

In order to attain the objects described above, according to the presentinvention, lectin for checking a blood type is extracted from seeds ofbitter gourd, balsam pear, or Momordica charantia. According to thepresent invention, a blood type checking reagent contains the lectin.The lectin extracted from seeds of bitter gourd preferably has amolecular weight between 100,000 and 170,000 measured withpoly-acrylamide gel electrophoresis in existence of sodium dodecylsulfate (SDS-PAGE).

According to the present invention, a method of checking a blood typeincludes the steps of: extracting lectin from seeds of bitter gourd,balsam pear, or Momordica charantia; preparing a blood type checkingreagent containing the lectin; and checking a blood type using the bloodtype checking reagent.

With the blood type checking reagent containing the anti-H lectin of thepresent invention, it is possible to accurately and quickly determine ablood type even from a small bloodstain.

In the present invention, the blood type checking reagent includes thelectin having high agglutinin tilter and extracted from seeds bittergourd, i.e., a low cost raw material. In general, after removing fleshfrom bitter gourd, seeds are usually disposed or used for feedinganimals. Therefore, cost of the low material is very low. Further, it ispossible to extract and refine the lectin with cost same as that ofconventional lectin. Accordingly, it is possible to produce the lectinwith low cost as compared with conventional lectin for checking a bloodtype.

Further, as compared with conventional lectin, the lectin extracted fromseeds of bitter gourd blood exhibits a specific agglutination reactionor hemagglutination relative to H-antigen and high agglutinin tilter.Accordingly, it is possible to accurately and quickly determine a bloodtype even from a small bloodstain, thereby obtaining reliable data forforensic investigation. Due to the high agglutinin tilter, the lectinextracted from seeds of bitter gourd shows stable agglutinin tilterwithin lot with little influence of raw materials.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(A) to 1(D) are schematic views showing results ofhemagglutination, wherein FIG. 1(A) shows hemagglutination of O-typeblood using a blood type checking reagent according to an embodiment ofthe present invention, FIG. 1(B) shows hemagglutination of O-type bloodusing a blood type checking reagent containing Ulex anti-H lectin, FIG.1(C) shows hemagglutination of Para-Bombay-type blood using the bloodtype checking reagent according to the embodiment of the presentinvention, and FIG. 1(D) shows hemagglutination of Para-Bombay-typeblood using the blood type checking reagent containing Ulex anti-Hlectin.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Hereunder, embodiments of the present invention will be explained withreference to the accompanying drawings. The explanation covers seeds ofbitter gourd, extraction and refinement of lectin, a range of molecularweight, blood type determination, a blood type checking reagent, and anelution test of bloodstain.

According to the present invention, a type of bitter gourd is notlimited to a specific one. It is possible to extract lectin from anytypes of bitter gourd, and seeds may be fully matured or pre-matured. Itis found that matured or ripe seeds provide lectin with higheragglutinin tilter. However, it is possible to obtain the same effect ondetermining a blood type with matured seeds or pre-matured seeds.

According to the present invention, the lectin preferably has amolecular weight between 100,000 and 170,000. The molecular weight maybe measured with poly-acrylamide gel electrophoresis in existence ofsodium dodecyl sulfate (SDS-PAGE), in particular, with non-continuousgel electrophoresis (Laemmli method) using a concentrated gel. It isfound that the lectin with a molecular weight between 100,000 and170,000 effectively agglutinates blood. That is, it is difficult toagglutinate blood with lectin having a molecular weight less than100,000 or larger than 170,000.

An operation of extracting the lectin will be explained next. Driedseeds of bitter gourd, regardless of matured or pre-matured seeds, areused.

1. 10 g of dried bitter gourd seeds were ground in a coffee mill toobtain fine power.2. The fine powder was added to 100 ml of distilled water, and themixture was stirred with a stirrer for three hours.3. The mixture was filtered with a bleached cloth to obtain filtrate.4. The filtrate was centrifuged with a centrifuge for 10 minutes at 5000rpm to obtain supernatant.5. 150 ml (1.5 times of the distill water added in step 2) of ethanolwas added to the supernatant, and the mixture was stirred and placedstill for one hour.6. Supernatant of the mixture was removed to obtain deposit, and thedeposit was centrifuged with a centrifuge for 10 minutes at 5000 rpm.7. Supernatant was removed to obtain deposit, and the deposit was spreadon an evaporating dish to be dried.8. The dried deposit was collected and ground with a mortar to obtainpowder (about 0.3 g). The powder was added to 5 to 10 ml of normalsaline solution containing gelatin to obtain solution.9. The solution was centrifuged with a centrifuge for 10 minutes at 5000rpm to obtain supernatant as anti-H lectin.

A method of measuring agglutinin tilter of the lectin thus obtained willbe explained next.

1. Serum was diluted double with normal saline solution, and dividedinto test tubes by 0.25 ml each.2. O-type blood cells corresponding to the anti-H lectin was washed withnormal saline solution three times to obtain 2% normal saline solutionsuspension. 0.25 ml of the normal saline solution suspension was addedto the diluted serum in a test tube to obtain a mixture. The test tubewas then rigorously shaken.3. The mixture was centrifuged with a centrifuge for 1 minute at 1000rpm.4. The test tube was gently shaken to observe hemagglutination.5. An extent of hemagglutination was classified from 1 to 3. Maximumdilution number of a test tube showing hemagglutination of 1 isdesignated as agglutinin tilter of the serum.

The extent of hemagglutination is measured relative to various types ofblood (refer to Table 4). For example, agglutinin tilter of O-type bloodmeasured with anti-H lectin extracted from seeds of bitter gourd wasfound to be 2⁸ (256) times with a hole-glass method (after placed in aroom temperature for 30 minutes) or 2⁹ (512) times with the test tubemethod (after centrifuged for 1 minute at 1000 rpm). The method ofmeasuring agglutinin tilter is disclosed in “forensic serology testmanual”, 90 page, table 3-12 (Published by Kinbara Shuppan).

A process of dissociation test and evaluating an agglutination reactionwill be explained next. When a bloodstain is attached to a piece ofmetal, a stone, a piece of plastic, or a piece of fiber, a cotton fiberor a small gauze dampened with distilled water is rubbed against alocation of the bloodstain, so that the bloodstain is stick to thecotton fiber or the gauze as a test specimen. Alternatively, thebloodstain is stick to a small amount of fibers solved in a solution,and the fibers are dried to obtain a test specimen. When it is difficultto cut out a portion with a bloodstain, or a bloodstain is stick topowder or a block, the same procedure is applied to obtain a testspecimen.

After obtaining a test specimen, a method described in “forensicserology test manual”, 181 page, table 4-22 (Published by KinbaraShuppan) is performed. The method has been frequently used in forensicinvestigation due to simplicity and high sensitivity. Apparatus andreagents used in the method include an environmental bath (Constanttemperature bath LT-480 manufactured by ADVENTEC), test tubes, holeglasses, anti-A serum, anti-B serum, anti-H lectin, respirator, andblood cell indicator.

1. A portion of a sample with bloodstain or a target portion was cut outin about 1.5 cm square, and divided into three pieces.2. Each of the three pieces was placed in a test tube with a specimenname (bloodstain, control) and a name of agglutinin (anti-H, anti-A,anti-B).3. 0.1 ml of agglutinin was added to the test tube with a correspondinglabel. Anti-H lectin was used as the anti-H agglutinin.4. The agglutinin absorbed a blood sample.5. After absorbing the blood sample, cold normal saline solution waspoured into the test tube in full. Then, normal saline solutioncontaining un-reacted antibody was removed while the specimen remainedin the test tube.6. The specimen dampened with normal saline solution in the test tubewas divided into three portions. 0.2% erythrocyte suspension of O-type,A-type, or B-type was added to each of the three portions, and mixtureswere heated at 56° C. for 10 minutes (dissociation).7. The test tube was centrifuged with a centrifuge for 1 minute at 1000rpm.

A method of evaluating an agglutination reaction or hemagglutination isperformed as follows with reference to “forensic serology test manual”,181 page, table 4-22 (Published by Kinbara Shuppan). After beingcentrifuged, the test tube is gently shaken, and the result is evaluatedbased on an evaluation table. In this case, the results ofhemagglutination are compared between the anti-H lectin from Ulex andthe anti-H lectin from bitter gourd.

In the embodiment, the anti-H lectin is extracted from seeds of bittergourd (balsam pear, or Momordica charantia), and is formed of ahemagglutination protein. Accordingly, in a case of Para-Bombay-typeblood, the anti-H lectin does not exhibit hemagglutination as shown inFIG. 1(C), indicating that the lectin from bitter gourd is an anti-Htype.

FIG. 1(A) shows hemagglutination of O-type blood using the blood typechecking reagent containing the anti-H lectin from bitter gourdaccording to the embodiment of the present invention, and FIG. 1(B)shows hemagglutination of O-type blood using a blood type checkingreagent containing Ulex anti-H lectin. FIG. 1(C) shows hemagglutinationof Para-Bombay-type blood using the blood type checking reagentaccording to the embodiment of the present invention, and nohemagglutination is shown. FIG. 1(D) shows hemagglutination ofPara-Bombay-type blood using the blood type checking reagent containingUlex anti-H lectin, and no hemagglutination is shown.

A process of extracting the lectin from bitter gourd will be explainednext.

1. 100 g of seeds of bitter gourd were ground in a coffee mill to obtainpowder.2. The powder was added to one litter of distilled water in a container,and the mixture was stirred with a stirrer.3. The mixture was placed still for more than 12 hours to extract seedcomponents of bitter gourd.4. The mixture was filtered with a bleached cloth to obtain extractedsolution.5. The extracted solution was centrifuged with a centrifuge for 30minutes at 3000 rpm to obtain supernatant.6. After being centrifuged, approximately two litters of ethanol wasadded to the supernatant, and the mixture was stirred and placed stillfor 12 hours at 4° c. for incubation.

Through the steps described above, it is possible to extract crudeanti-H lectin. Although the crude anti-H lectin contains lectin, otherproteins, and sugar, it is found that the crude anti-H lectin exhibitssufficient agglutinin tilter.

A process of refining the crude anti-H lectin thus obtained will beexplained next.

1. Supernatant of the extracted fluid was removed with an aspirator toobtain deposit.2. The deposit was centrifuged with a centrifuge for 30 minutes at 3000rpm to separate a liquid portion.3. The deposit was spread in a wide mouth container and dried to obtaina dried product.4. The dried product was solved in 100 ml of normal saline solutioncontaining gelatin to obtain solution.5. The solution was centrifuged with a centrifuge for 30 minutes at 3000rpm to obtain supernatant.

Through the steps described above, it is possible to obtain refinedanti-H lectin. The lectin thud refined has a molecular weight of about120,000 measured with the SDS-PAGE method. The anti-H lectin thusrefined contains a less amount of other proteins and sugar, and has ahigher purity and agglutinin tilter.

An experiment of determining a blood type using a blood type checkingreagent containing the lectin extracted from bitter gourd describedabove will be explained next. The experiment includes a test ofdetermining a type of a blood cell through hemagglutination (chart testor absorption-elution test) using the anti-H lectin extracted frombitter gourd; a dissociation test of an ABO type blood test ofbloodstain using the anti-H lectin extracted from bitter gourd; and adissociation test of an Para-Bombay-type blood test of bloodstain usingthe anti-H lectin extracted from bitter gourd through the methoddescribed above.

Results of the tests using 50 μl of the lectin are shown in thefollowing Table 1 to Table 3.

TABLE 1 Chart test using the bitter gourd anti-H lectin Bitter gourdBitter gourd Bitter gourd lectin lectin lectin Chart Test (agglutinin(agglutinin (agglutinin Result tilter × 32) tilter × 64) tilter × 128)Para-Bombay- — ‡* ‡* type blood cell (no H-antigen) O-type blood 3 3 3cell ‡*: abnormal hemagglutination due to excess antigen

TABLE 2 Dissociation test of the ABO type blood test of a bloodstainusing the bitter gourd anti-H lectin Bitter Bitter Bitter gourd gourdgourd Anti-B Ulex lectin lectin lectin lectin Anti-A antigen (agglutinin(agglutinin (agglutinin (agglutinin Specimen antigen (α) (β) tilter ×32) tilter × 32) tilter × 64) tilter × 128) A-type 2 — —* — 1 2 bloodcell A-type — 2 —* ‡ 2 3 blood cell A-type — — —* ‡ 2 3 blood cell*Undetectable with the anti-H lectin extracted from Ulex seeds

TABLE 3 Dissociation test of a bloodstain using the bitter gourd anti-Hlectin Bitter gourd lectin Specimen Time ×32 ×64 ×128 ×256 ×512 ×1024A-type 10 ‡ 1 1 1 1 1 bloodstain A-type 10 1 2 2 2 2 2 bloodstain Para-10 — — — — — — Bombay bloodstain A-type 15 ‡ 2 2 2 2 2 bloodstain A-type15 2 3 3 3 3 3 bloodstain Para- 15 — — — — — — Bombay bloodstain A-type20 ‡ 2 2 2 2 2 bloodstain A-type 20 2 3 3 3 3 3 bloodstain Para- 20 — —— — — — Bombay bloodstain Para- 30 — — — — — — Bombay bloodstain

As shown in Table 1 to Table 3, the blood checking reagent containingthe anti-H lectin extracted from bitter gourd exhibited high reactivityrelative to a bloodstain, while the conventional Ulex lectin did notdetect. Accordingly, the blood checking reagent of the present inventionis superior to the conventional reagent in terms of agglutinin tilterand hemagglutination.

Results of an evaluation of the blood checking reagent of the presentinvention will be explained with reference to Table 4 to Table 8.

TABLE 4 Agglutinin tilter relative to various blood cells Agglutinintilter Blood cell Hole glass Test tube Agglutinin (2% suspension) method(1) method (2) Anti-H lectin O-type blood 2⁸ (256) 2⁹ (512) extractedfrom cell seeds of bitter A₁-type blood 2⁶ (64)  2⁷ (128) gourd cellA₂-type blood 2⁸ (256) 2⁹ (512) cell B-type blood 2⁶ (64)  2⁷ (128) cell(1) Evaluate at room temperature for 30 minutes (2) Centrifuged at 1000rpm for 1 minutes

TABLE 5 Agglutinin tilter (chart test: mixed on a flat glass plate)Anti-H lectin Anti-H lectin Blood cell extracted from seeds extractedfrom (2% suspension) of bitter gourd seeds of Ulex O-type blood cell Ca.20 seconds Ca. 2 minutes A₁-type blood cell Ca. 35 seconds Ca. 15minutes A₂-type blood cell Ca. 25 seconds Ca. 2 minutes B-type bloodcell Ca. 35 seconds Ca. 15 minutes Para-Bombay-type No hemagglutinationNo blood cell hemagglutination

As shown in Table 5, the conventional anti-H lectin extracted from seedsof Ulex took 2 to 15 minutes for hemagglutination. On the other hand,the anti-H lectin extracted from seeds of bitter gourd took few tens ofseconds for hemagglutination, indicating high reactivity.Para-Bombay-type blood cell, occurring one out of million people, didnot show hemagglutination, indicating the lectin extracted from seeds ofbitter gourd is the anti-H lectin.

TABLE 6 Storage stability (relative to O-type blood cell) Lot. No.GL4917-1 Storage temperature Storage time Agglutinin tilter   20° C. 3months 2⁸ (256)    4° C. 3 months 2⁸ (256) −30° C. 3 months 2⁸ (256)−80° C. 3 months 2⁸ (256)

As shown in Table 6, when the anti-H lectin was stored for 3 months,temperature did not affect the stability of agglutinin tilter.

TABLE 7 Stability during heating (relative to O-type blood cell) Heatingtemperature Storage time Agglutinin tilter 37° C. 60 minutes 2⁸ (256)55° C. 10 minutes 2⁸ (256) 56° C. 30 minutes 2⁷ (128) 60° C. 10 minutes2⁷ (128) 70° C. 10 minutes 2³ (8) 

As shown in Table 7, when the anti-H lectin was heated, high agglutinintilter (256) was maintained up to 55° C.

TABLE 8 Dissociation test of various blood types of bloodstains(bloodstains on cotton fibers: evaluated with 0.3% human blood cell)Anti-A Anti-B Bitter antigen antigen gourd Bloodstain (α) (β) lectinUlex lectin A-type 2 — 2 — bloodstain B-type — 2 3 — bloodstain O-type —— 3 — bloodstain Para- — — — — Bombay-type bloodstain

As shown in Table 8, it is possible to determine A-type bloodstain withanti-A antigen and the bitter gourd lectin (anti-H antigen), and B-typebloodstain with anti-B antigen and the bitter gourd lectin (anti-Hantigen). Further, it is not possible to determine O-type bloodstainwith anti-A antigen, anti-B antigen, and the conventional Ulex lectin.Accordingly, the blood checking reagent containing the anti-H lectinextracted from bitter gourd exhibited high reactivity in terms ofagglutinin tilter and hemagglutination, and was superior to theconventional reagent.

TABLE 9 Comparison of agglutinin tilter between the bitter gourd anti-Hlectin and commercially available anti-H lectin (relative to O-typeblood cell) Agglutinin tilter Price Hole Test (Japanese glass tubeAgglutinin Manufacture Yen) method (1) method (2) Bitter gourd 2⁸ (256)2⁹ (512) lectin Ulex Anti-H Honen Corp. 10,000 2⁶ (64)  2⁷ (128) lectinlectin (8 ml) Anti-H Honen Corp. 10,000 2⁸ (256) 2⁹ (512) lectin strong(2 ml) Anti-H Wako Pure 10,000 2³ (8)  2⁴ (16)  lectin Chemical (3 ml)Anti-H EY 10,000 2³ (8)  2⁴ (16)  lectin Laboratory (2 ml) (1) Evaluateat room temperature for 30 minutes (2) Centrifuged at 1000 rpm for 1minutes

As shown in Table 9, the blood checking reagent containing the anti-Hlectin extracted from bitter gourd exhibited high agglutinin tilter ascompared with the conventional reagents containing Ulex lectin.

While the invention has been explained with reference to the specificembodiments of the invention, the explanation is illustrative and theinvention is limited only by the appended claims.

1. A blood type checking reagent, comprising lectin extracted from seedsof bitter gourd (Momordica charantia) through the steps of: grinding theseeds of bitter gourd to obtain first power; processing the first powderto obtain second powder; adding the second powder to normal salinesolution containing gelatin to obtain a mixture; and centrifuging themixture to obtain supernatant as the lectin.
 2. The blood type checkingreagent according to claim 1, further comprising the steps of: addingthe first powder into distilled water to obtain a first mixture;filtering the first mixture to obtain filtrate; centrifuging thefiltrate to obtain first supernatant; adding the first supernatant toethanol to obtain a second mixture; removing second supernatant of thesecond mixture to obtain first deposit; centrifuging the first depositto obtain second deposit; and drying the second deposit to obtain thesecond powder.
 3. The blood type checking reagent according to claim 1,wherein said lectin has a molecular weight between 100,000 and 170,000measured with poly-acrylamide gel electrophoresis in existence of sodiumdodecyl sulfate (SDS-PAGE).